Ivan Černý

Steroid-porphyrin conjugate for saccharide sensing in protic media.

A new saccharide receptor in protic media has been designed and synthesized. The receptor combines advantages of steroids, which are responsible for saccharide binding, and of the porphyrin moiety acting as a signalling component of the molecule due to changes in UV-vis electronic spectra. The synthesis is based on condensation of steroid aldehyde with pyrrole to form the porphyrin unit with four protected steroid moieties. After deprotection, meso-substituted porphyrin contains 12-hydroxy groups on the steroidal part. The receptor is soluble in aqueous solutions and exhibits high complexation affinity towards saccharides. Because the receptor extensively aggregates in water, most of the experiments were performed in 50% aqueous 2-propanol where aggregation is significantly eliminated. Binding is evidenced by spectral changes in the Soret region of the receptor in UV-vis absorption spectra allowing the evaluation of the binding constants. Additional confirmation of binding is obtained using 1H NMR, Raman and IR spectroscopies and the surface plasmon resonance technique. The receptor exhibits higher selectivity for oligosaccharides over monosaccharide. The results point to the importance of a combination of multiple binding via H-bonding and hydrophobic interactions.

Metal coordination as a tool for controlling the self-assembling and gelation properties of novel type cholic amide–phenanthroline gelating agent

Abstract (3α,7α,12α)-Trihydroxy-N-[1,10-phenanthrolin-5-yl]-5β-cholan-24-amide was found to be a powerful gelating agent for methanol–water in gelator to solvent ratio starting from 0.1% in absence of metal ion. Formation of phenanthroline–zinc (II) 2:1 complex changes dramatically gelating properties; when stored, it dissolves into clear solution without Tyndall effect and this solution, when heated to ca. 70°C reversibly forms a gel again, without chemical change as proven by NMR spectrometry. Structures of the gels of cholic amide–phenanthroline and its Zn2+ complex were studied by SEM.

Immunoanalysis of isoflavonoids in Pisum sativum and Vigna radiata

Abstract Radioimmunoassays (RIAs) combined with liquid chromatography were used for study of isoflavonoids in seeds of pea Pisum sativum and mung bean Vigna radiata. Radioimmunoassays with these specifities were used: (1) daidzein and its 4′-derivatives (e.g. formononetin); (2) daidzein and its 7-derivatives (e.g. daidzin, isoformononetin); (3) genistein and its 4′-derivatives (e.g. biochanin A); and (4) genistein and its 7-derivatives (e.g. genistin, prunetin). Dormant or germinating seeds were extracted with 80% ethanol. Immunoreactivities were measured either in crude extracts or after chromatographic fractionation by HPLC (reversed phase, octadecylsilica). Chromatographic mobilities of immunoreactive fractions were compared to those of daidzein, daidzin, formononetin, isoformononetin, genistein, genistin biochanin A and prunetin standards. Extracts from Vigna radinfa contained daidzein, genistein and their 7- O glucosides, daidzin and genistin, respectively. No immunoreactivity was recorded in HPLC fractions corresponding to glycosides in extracts from P. sativum, but the methods sensitive to 7-derivatives of daidzein and genistein showed peaks with chromatographic mobilities identical to those of the 7-methoxyderivatives, isoformononetin and prunetin, respectively. In additional experiments, the pea extracts were fractionated either by thin layer chromatography (TLC) on silica or by ion-exchange TLC on aminosilica. Identity of the daidzein-7 and genistein-7 immunoreactive entities with isoformononetin and prunetin, respectively, was confirmed by the identical chromatographic behavior in all these different chromatographic systems.

The identification and simultaneous quantification of 7-hydroxylated metabolites of pregnenolone, dehydroepiandrosterone, 3β,17β-androstenediol, and testosterone in human serum using gas chromatography–mass spectrometry

7-Hydroxy-metabolites of dehydroepiandrosterone (DHEA) and 3beta,17beta-androstenediol (AD) possess immunomodulatory and neuroprotective properties; therefore, the measurement of these steroids in patients with autoimmune diseases or disturbances in the CNS may be of interest. A gas chromatography-mass spectrometry (GC-MS) method for the determination of 7-hydroxy-metabolites of pregnenolone, DHEA, AD, and testosterone including the parent steroids was applied to serum samples from 12 adult men (27-66 years), 13 male adolescents (13-20 years), 5 boys (6-10 years), 15 women in the follicular phase of the menstrual cycle (22-45 years), 17 women in the luteal phase (22-45 years), and 4 girls (6-10 years). The steroids were age and sex dependent, but independent of the menstrual cycle. The ratio of the 7alpha-hydroxy-metabolites to their parent steroids were age dependent, exhibiting an increasing trend (p < 0.0001, ANOVA) from pregnenolone (5%) to AD (20%). The ratio of 7beta- to 7alpha-metabolites ranged from 0.6 to 1. These results are consistent with models suggesting 7alpha-hydroxylation of the parent steroid, conversion to a 7-oxo-steroid and finally to the 7beta-hydroxylated-metabolite. Partial correlations suggested that 7-hydroxylation might reduce the concentration of circulating androgens. Despite the three times lower concentration of AD-metabolites, their antiglucocorticoid, immunomodulatory, and neuroprotective effects may be comparable to that of DHEA based on their reported greater biological activity.

Stereoselectivity of sodium borohydride reduction of saturated steroidal ketones utilizing conditions of Luche reduction.

A series of keto steroids were reduced with sodium borohydride in the presence of cerium(III) chloride or samarium(III) iodide (Luche reduction). The ratios of axial and equatorial alcohols were determined by HPLC and the results were compared with those obtained by a standard sodium borohydride reduction. The best results were obtained with the 2-keto derivative 1, 7-keto derivatives 5 and 6, and 12-keto derivative 8 where the cerium(III) ion addition resulted in the inversion of the axial/equatorial ratios. The Luche reduction of the 20-keto derivative 11 improved the proportion of the (20S)-alcohol in a mixture of (20S)/(20R) alcohols up to 35% from 5% in a standard sodium borohydride reduction.

Novel Deep Cavity Calix[4]pyrroles Derived from Steroidal Ketones

Novel steroidal calix[4]pyrroles were prepared in excellent yields from commercially available cholic acid derivatives using an efficient synthetic sequence. Once in hand, it was found that these calix[4]pyrroles exist in the form of four different configurational isomers. Separation of these isomers was achieved readily using normal phase HPLC techniques. Once purified, the steroidal calix[4]pyrroles were screened via -FABMS analyses to judge their utility in effecting the enantioselective recognition of appropriate organic anions. Results that provided support for antipodal R > S selectivity were obtained in the case of both tartaric acid and mandelic acid. Direct extraction studies were then carried out and these confirmed the pattern of R > S selectivity observed by -FABMS.

Preparation of 2-amino-1,6-anhydro-2,3-dideoxy-β-d-arabino-hexopyranose. 1H- and 13C-n.m.r. spectra of deoxy derivatives of 2-amino-1,6-anhydro-2-deoxy-d-glucose and 2-amino-1,6-anhydro-2-deoxy-d-mannose

Partial benzyloxycarbonylation of 1,6-anhydro-d-mannosamine, subsequent mesylation, and treatment with sodium methoxide gave 1,6:3,4-dianhydro-2-benzyloxycarbonylamino-2-deoxy-β-d-altropyranose (9). Oxirane-ring cleavage in 9 with potassium iodide, acetylation, and dehalogenation with tributylstannane, gave 4-O-acetyl-1,6-anhydro-2-benzyloxycarbonylamino-2,3-dideoxy-β-d-arabino-hexopyranose (13). Removal of protective groups in 13 by deacetylation and hydrogenolysis gave the hydrochloride of the title compound (15). 2-Amino-1,6-anhydro-2,3-dideoxy-β-d-ribo- (18) and -2,4-dideoxy-β-d-xylo-hexopyranose hydrochlorides (21) were prepared from the corresponding azido derivatives. 1H-and 13C-n.m.r. spectra of 15, 18, 21, and of 2-amino-1,6-anhydro-2,4-dideoxy-β-d-lyxo-hexopyranose hydrochloride in D2O were compared with those of the corresponding deoxy derivatives and other related compounds. Isomerization of 2-amino-1,6:3,4-dianhydro-2-deoxy-β-d-altropyranose into the 2,3-epimine was also studied.

Syntheses of 19-[O-(carboxymethyl)oxime] haptens of epipregnanolone and pregnanolone

O-(Carboxymethyl)oximes 1 and 2 derived from two epimeric 5beta-pregnanolones (3beta-hydroxy-5beta-pregnan-20-one and 3alpha-hydroxy-5beta-pregnan-20-one) in position 19 were prepared. Two synthetic routes were employed, both using protection of the 20-keto group after reduction into the (20R)-alcohol in the form of acetate. In the first route, (20R)-19-hydroxy-5beta-pregnan-3beta,20-diyl diacetate (3) was transformed into the corresponding 19-[O-(carboxymethyl)oxime] methyl ester 6, then deacetylated by acid and partially silylated with tert-butyldimethylsilyl chloride. The desired 3-O-silylated derivative 8 was separated, oxidized to the 20-ketone and protecting groups were sequentially removed to give the first title hapten 1. The second route started from (20R)-19-hydroxy-3-oxopregn-4-en-20-yl acetate (11), which was hydrogenated in the presence of base to the 5beta-pregnan-3-one derivative 12, protected in position 19 with tert-butyldimethylsilyl group and reduced with borohydride. The prevailing 3alpha-alcohol 15 was separated, protected in position 3 with a methoxymethyl group, deprotected in position 19 and transformed into the 19-[O-(carboxymethyl)oxime] 19. After deacetylation, esterification with diazomethane and oxidation in position 20, the pregnanolone skeleton was regenerated. Final deprotection steps gave the second title hapten 2. Both haptens, i.e., (19E)-3beta- and -3alpha-hydroxy-20-oxo-5beta-pregnan-19-al 19-[O-(carboxymethyl)oxime], were designed for the development of immunoassays of the corresponding parent neuroactive steroids.

Synthesis of [19- 2H3]-analogs of dehydroepiandrosterone and pregnenolone and their sulfates.

Deuterated analogs of pregnenolone and pregnenolone sulfate with three atoms of deuterium in position 19 were prepared. The synthetic approach was developed on derivatives of dehydroepiandrosterone, where initial intermediates were well characterized, and then applied to the pregnenolone series. Starting 19-hydroxy compounds were transformed into 3alpha,5-cycloderivatives to simplify the Jones oxidation into the corresponding 19-oic acids. After oxidation, rearrangement to 3-hydroxy-5-enes, and suitable protection, two deuterium atoms were introduced by lithium aluminum deuteride reduction. Mesylate exchange by iodide in the presence of zinc and deuterium oxide added third deuterium atom. Deprotection gave title analogs with about 93-95% content of d3-derivative, the rest was mainly not fully deuterated d2-analogue as followed from the mass spectra analysis. Thus, 3beta-hydroxy[19-2H3]androst-5-en-17-one was prepared in 14 steps from 19-hydroxy-17-oxoandrost-5-en-3beta-yl acetate in 8.9% yield, the analogous sequence in the pregnenolone series gave 3beta-hydroxy[19-2H3]pregn-5-en-20-one in 7.3% yield. Corresponding sulfates were prepared via pyridinium salts in 53 and 57% yields, respectively. Fully assigned NMR data of selected pregnenolone derivatives were given.

Synthesis of two new haptens of 16α-hydroxydehydroepiandrosterone (3β,16α-dihydroxyandrost-5-en-17-one)

Synthetic routes leading to 19E and 7Z O-(carboxymethyl)oximes derived from 16α-hydroxydehydroepiandrosterone were developed using two independent methods for introduction of the 16α-hydroxy group. Firstly, the oxime moiety was built, and then, either epoxidation of the enol acetate followed by the boron trifluoride mediated rearrangement or alkaline hydrolysis of the corresponding α-bromide in aqueous N,N-dimethylformamide were employed. The last step in both methods was removal of the protecting groups, which consisted of acid deprotection of the acetates and gentle alkaline hydrolysis of the methyl ester. Final haptens were designed as components for immunoanalytical kits.